Recently, we have identified a large polytopic membrane protein, NPC1L1 that shares significant homology (42% amino acid identity) with the Niemann-Pick C1 (NPC1) disease protein. NPC1 disease is a severe lysosomal lipidosis in which the egress of cholesterol and other lipids from the endosomal/lysosomal (E/L) system is defective, leading to neurodegeneration and premature demise. The proteins responsible for the two forms of NPC disease, NPC1 and NPC2, were recently identified. Preliminary characterization of these proteins suggests that NPC1 may act as a lipid permease on the membranes of late endosomes, whereas NPC2 is a small, soluble, cholesterol-binding lysosomal protein. The role of our newly identified NPC1 homologue, NPC1L1, is currently unknown and no diseases have been ascribed to its loss of function. Based on the homology between NPC1 and NPC1L1 and our preliminary data, we hypothesize that NPC1L1 has a lipid permease function similar to that of NPC1 but resides in a different subcellular location. Therefore, the overall objectives of the proposed research are to characterize the function of NPC1L1 and determine its role in subcellular lipid and/or cholesterol transport. Efforts will first be directed towards the analysis and characterization of the topology and intracellular location of the NPC1L1 protein to determine the cellular location in which it functions. Solution of its membrane topology will establish the direction of its potential pump activity and also its relationship to the newly identified resistance-nodulation-division (RND) family of eukaryotic permeases. Next, analysis and characterization of the function(s) of the NPC1L1 protein and its potential regulation by subcellular cholesterol and or lipid levels will be carried out to determine whether NPC1L1 exhibits a fatty acid, or other lipid, permease activity. These studies will be accomplished by expression of NPC1L1 in prokaryotes engineered to contain mutations in their endogenous RND permease genes relevant to our studies. Expression in yeast and mammalian cells will also be utilized, as needed, to further characterize the function(s) of this protein. Finally, generation and characterization of an NPC1L1 knockout mouse model should provide us with the necessary data to completely characterize the physiological function of NPC1L1 and its involvement in lipid/cholesterol transport or homeostasis.